According to our knowledge, we provided the first photographs and study of the female reproductive organs of the African Monarch D. chrysippus. Consistent with early schematic drawings by Mal et al. 30, female D. chrysippus butterflies present two signa. Each signum are of similar size, of a beaver-tail shape in mated females, and cover on average 32% of each side of the corpus bursa, but do not extend into the appendix bursa.
Morphological traits such as the count and shape of the signa and the smoothness or complexity of their surface vary enormously among Lepidoptera species, and could provide taxon-specific diagnostic characters 31. The signa have for example been described as smooth, or ornamented with micro-protuberances of different ornamented shapes ie. spikes, teeth, spines, horns, bands, patches, or plates, 25. Mal et al. 30 did not provide any details on the signum structures in D. chrysippus, but we showed that both signa are covered with spike-like sclerotized structures of similar size. They give the signa their darker colour compared to the rest of the bursa. In comparison, the Monarch butterfly, D. plexippus (Linnaeus, 1758), has a large, pear-shaped corpus bursa with two large signa, each covered with bands of heavily chitinized micro-protuberances pointed in opposite directions from the median 32. These spike-like structures are illustrated in detail in Rogers & Wells 33. The female organs of D. plexippus thus seem similar to those we described in D. chrysippus, however in general such bisignate condition is uncommon in Lepidoptera. For example, the South-East Asian D. genutia butterfly (Cramer, 1779) has similarly shaped corpus bursa than D. chrysippus, but only present one signum, which is described as rod-shaped 30. In geometrid moths, the two signa character is also rare 34 and references therein. However, the lack of extensive morphological revision describing the female genitalia of Nymphalidae or Danainae butterflies, where D. chrysippus is classified, does not currently allow the use of these morphological results in a wider evolutionary framework for these butterflies, contrasting for instance by the work done in Tortricidae 35.
The signum structures coupled with muscles associated with the corpus bursa 36 have been described as ‘lamina dentata’ 37 a structure possibly involved in the digestion, by grating, of the nutrient-rich surface of the spermatophores after copulations 4,25,38. If this is true, we expect that natural selection will act on the female genitalia in response to sexual conflict. We hypothesised two scenarios during which female organs might evolve to due to changes in the size of the male’s contributions to copulation between populations, and in order to optimize the digestion of the male’s spermatophore:
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In scenario (I), females receive larger, or many spermatophores in their lifetime. As larger spermatophore might act as mating plugs 39,40, females would evolve organs to efficiently digest each nuptial gift to be able to remate and avoid the fertilization of all their eggs by one unique genitor.
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In scenario (II), females receive small or few spermatophores in their lifetime. As the nutrients received from the males can be upcycled towards the production of eggs, or of better-quality eggs 41, females would evolve organs to optimise the intake from the small nuptial gift 42.
Unfortunately, our sample size was too small to support significant differences in the spermatophores size between the populations of D. chrysippus. There was however evidence in other butterfly species, that spermatophores are smaller as the sex-ratio distortion increased in the host populations. For example, in the blue-moon butterfly, H. bolina, Charlat et al 24 suggested that males become resource depleted in populations where the MK-symbiont is common. Similar studies in D. chrysippus, and other Lepidoptera, will be needed to confirm whether male resource depletion is common in MK-infected species. Nonetheless, we found that the surface area of the corpus bursa, that of the signa, and their ratio, were different between populations of D. chrysippus. Particularly, in Spiroplasma-free populations from South Africa with no sex-ratio distortion, the ratio between signum and bursa area is intermediate. In contrast, in the Kenyan and Rwandan populations showing female-biased or variable sex-ratio distortion levels, respectively (with high and medium prevalence of the MK-Spiroplasma symbiont, respectively), the ratio between the signum and bursa area is either intermediate to large, or intermediate to small, respectively. Such pattern may be suggesting selection in the sex-ratio distorted populations. Altogether, our results might best support the second scenario presented above: in D. chrysippus, females from highly female-biased populations presented a larger area of their small corpus bursae being covered with signa structures. Such larger structure is likely to efficiently mechanically digest the small spermatophores these females receive from males. However, the cofounding effect of population needs to be further investigated. Similarly, although it has been suggested from few other Lepidoptera species 38, whether and how the digestion of the spermatophore occurs, and whether the signa and bursa are indeed involved in the mechanical digestion of the spermatophore in D. chrysippus remains to be fully experimentally tested.
Finally, we demonstrated that in D. chrysippus the size of the corpus bursa, that of the signa, and their ratio, varied with the mating status of the females regardless of their population of origin. In each population, the virgin females showed smaller organs, while mated females showed expanded organs. Comparative illustrations of virgin versus mated female genitalia are scarce in insects and other arthropods 43,44, but there is evidence for the female organs to vary in their shape, size and possible functionality after mating. For example, in seed beetles (Coleoptera: Bruchidae), male genitalia are armed with sclerotized spikes that serve as anchors to the female during copulation but that cause scar-tissues to be observed in mated females only 45,46. Similarly, in the orb-weaving spider, Larinia jeskovi, the male removes a coupling device (ie. scapus) from the female external genitalia after copulation, inhibiting the possibility for the female to remate 44. In D. chrysippus, we suggest that mated females showed larger organs because they were likely filled up with the spermatophores. Unfortunately, we should note that the KOH treatment for the fixation of the female organs destroyed the spermatophores within the bursae in D. chrysippus. Such inconvenience does not allow (I) to precisely determine which females were mated or not before dissection, although lab-reared virgin individuals were informative, (II) to determine how many times each female had mated before dissection, and (III) to evaluate the size and composition of the male’s contribution to mating in each population. This has challenged our ability to obtain both the data on males’ contribution and female genitalia traits from the same individuals, which would be of importance for further study of the male-female conflicts that occur in this and other species.